Journal: bioRxiv

Article Title: Network-based prioritisation and validation of novel regulators of vascular smooth muscle cell proliferation in disease

doi: 10.1101/2023.08.25.554834

Figure Lengend Snippet: (A) Predicted inputs to TIMP1 in the PrP GRN, coloured as described in left panel of . (B) Representative immunohistochemistry image for αSMA (blue) and TIMP1 (brown) in non-plaque human aorta (N=7), scale bar=500 µm (overview) and 100 µm (zoomed view). (C) Enrichment plots from gene set enrichment analysis (GSEA) for E2F targets (p adj = 0.005) and G2M checkpoint genes (p adj = 0.04) in bulk RNA-seq data from human VSMCs (hVSMCs) treated with 500 ng/mL recombinant human (rh) TIMP1 for 6 hours versus control cells (N=6 independent hVSMC isolates). (D) Proportion of EdU+ hVSMCs following 16 hours EdU incorporation in cells treated with 500 ng/mL rhTIMP1 or vehicle controls (CTRL), standardised to the average of all samples (N=6 independent hVSMC isolates). (E) Representative images of Myh11-Confetti VSMCs isolated from murine aortas and cultured for 21 days in the presence of vehicle control, 500 ng/mL recombinant murine TIMP1 or 2 ng/mL PDGF-BB. Scale bar=500 µm. (F, G) Quantification of number and size of clonally expanded patches of Confetti+ VSMCs over 21 days of culture. Statistical significance assessed via a generalised linear model, N=4 animals.

Article Snippet: TIMP1-containing fractions were identified by Western blot analysis using the anti-TIMP1 antibody (human: rabbit anti-human antibody; 1:1,000, cat. #8946; Cell Signaling Technology, mouse: goat anti-murine antibody 1:1,000, cat. #AF980; Biotechne R&D biosystems), pooled and concentrated by centrifugal filter (Amicon Ultra 15, 10 kD; Merck).

Techniques: Immunohistochemistry, RNA Sequencing, Recombinant, Control, Isolation, Cell Culture

Journal: bioRxiv

Article Title: Network-based prioritisation and validation of novel regulators of vascular smooth muscle cell proliferation in disease

doi: 10.1101/2023.08.25.554834

Figure Lengend Snippet: (A) Proteome profiler phosphokinase array of human VSMCs (hVSMCs) following 15 minutes treatment of 500 ng/mL recombinant human (rh) TIMP1 or vehicle control. Quantification of relative spot intensity by densitometry. (B) Western blot of total, pS727 and pT705 STAT3 in serum starved hVSMCs analysed 0, 5 10, 15 or 30 minutes after rhTIMP1 treatment. (N=5 hVSMC isolates). (C) Quantification of relative band intensity, normalised to total STAT3 levels (N=5 hVSMC isolates), statistical significance determined using ANOVA. (D) ChIP qPCR analysis of STAT3 binding at STAT3 targets ( TWIST and JUN ) and a negative control ( AMICA1 ) gene promoters, in serum starved control hVSMCs and 15 minutes following 500 ng/mL rhTIMP1 treatment. Graph shows anti-STAT3 and control IgG precipitated DNA as a percent of input. N=3 hVSMC isolates. (E) Representative images of STAT3 and Ki67 staining in serum starved control hVSMCs and after 15 mins 500 ng/mL rhTIMP1 treatment (N=3 hVSMC isolates analysed in triplicate). Arrowheads indicate KI67+ cells. Scale bar=50 µm. (F) Quantification of relative fluorescence intensity of nuclear pSTAT (S727) staining in panel E, for KI67+ and KI67-cells. (G) Representative images of VSMCs isolated from Myh11-Confetti aortas cultured 21 days in +/− 500 ng rhTIMP1 and/or 10 uM TT101 (N=6). Scale bar=500 µm. (H) Number and size of clonally expanded patches formed by lineage labelled VSMCs from Myh11-Confetti aortas, treated as indicated and imaged over 21 days. Q uantified using ImageJ. Statistical significance assessed via generalised linear model. N=6. Asterisk indicates p<0.05.

Article Snippet: TIMP1-containing fractions were identified by Western blot analysis using the anti-TIMP1 antibody (human: rabbit anti-human antibody; 1:1,000, cat. #8946; Cell Signaling Technology, mouse: goat anti-murine antibody 1:1,000, cat. #AF980; Biotechne R&D biosystems), pooled and concentrated by centrifugal filter (Amicon Ultra 15, 10 kD; Merck).

Techniques: Recombinant, Control, Western Blot, ChIP-qPCR, Binding Assay, Negative Control, Staining, Fluorescence, Isolation, Cell Culture

Journal: bioRxiv

Article Title: Network-based prioritisation and validation of novel regulators of vascular smooth muscle cell proliferation in disease

doi: 10.1101/2023.08.25.554834

Figure Lengend Snippet: (A) Number and size of clonally expanded patches formed by lineage labelled VSMCs from Myh11-Confetti animals, treated with 500 ng/mL recombinant TIMP1 or equimolar amount of N-TIMP1 tracked over 21 days of culture. Statistical significance assessed via generalised linear model. N=4. (B) Representative image of CD74 immunostaining (magenta) in lineage labelled mouse carotids 10 days post ligation of Myh11-Confetti animals. Magnified panels also show DAPI counterstained nuclei (top: merge of Confetti signals, CD74 and DAPI, middle: DAPI and RFP, lower: DAPI and CD74). White pointers marks CD74/RFP double positive cells. N=5 animals. Scale bar=100 µm (overview), 30 µm (zoomed view) (C) Representative image of murine VSMCs treated with 500 ng/mL recombinant murine (rm) TIMP1 four days post isolation and immunostained for pSTAT3 (S727, red) and CD74 (green). Nuclei are counterstained with DAPI (blue). Red pointer marks a STAT3 high cells, arrowhead points to a STAT3 low cell in merged and individual channels. (D) Quantification of cellular CD74 levels in panel C, stratified by high or low nuclear pSTAT3 intensity. N=4 animals, 4 repeats of each. Statistical significance was determined via student t-test. (E) Number and size of clonally expanded patches formed by lineage labelled VSMCs from Myh11-Confetti animals, treated with 500 ng/mL rmTIMP1 and/or a CD74 blocking antibody or peptide. (F) Representative western blot of serum started control human VSMCs (hVSMCs, UT), and cells treated for 5 minutes with 500 ng/mL recombinant human TIMP1 with or without +/− pre-treatment with an antibody (CD74 ab) or a peptide (CD74 pep) that blocks CD74. ( G ) Densitometric quantification of panel F. N=4 hVSMC isolates. ( H ) Representative immunohistochemistry image of CD74 in non-plaque human aorta. αSMA = blue, CD74 = brown. Scale bar=500 µm (overview) and 100 µm (zoomed view). N=7. (I) Representative images of RNA in situ hybridization for ACTA2 (green), TIMP1 (blue) and CD74 (red) in healthy human aorta, and plaque containing carotid. Blue arrows denote TIMP1 / ACTA2 + cells, red arrows denote CD74 / ACTA2 +cells. ( J ) Quantification of the % of ACTA2 positive cells also expressing TIMP1 or CD74 in the medial layer of non-plaque aorta (Healthy media) or in medial (Media Under Plaque) or Plaque regions of carotid endarterectomy samples (N=4 for each condition).

Article Snippet: TIMP1-containing fractions were identified by Western blot analysis using the anti-TIMP1 antibody (human: rabbit anti-human antibody; 1:1,000, cat. #8946; Cell Signaling Technology, mouse: goat anti-murine antibody 1:1,000, cat. #AF980; Biotechne R&D biosystems), pooled and concentrated by centrifugal filter (Amicon Ultra 15, 10 kD; Merck).

Techniques: Recombinant, Immunostaining, Ligation, Isolation, Blocking Assay, Western Blot, Control, Immunohistochemistry, RNA In Situ Hybridization, Expressing